Methods for enhancing the production of viral vaccines in PKR-deficient cell culture

ABSTRACT

Methods for enhancing the production of viral vaccines in animal cell culture are described. These methods rely on the manipulation of the cellular levels of certain interferon induced antiviral activities, in particular, cellular levels of double-stranded RNA (dsRNA) dependent kinase (PKR), PKR-deficient cells are obtained by a) transfecting a parent cell line with a PKR antisense polynucleotide; b) unaided uptake into a cell line by culturing said cell line in the presence of a PKR antisense polynucleotide; c) transfection of a parent cell line with a PKR dominant negative mutant; or d) culturing a cell line in the presence of 2-aminopurine. In cell cultures deficient for PKR, viral yield is enhanced by several orders of magnitude over cell cultures with normal levels of these proteins making these cell cultures useful for the production of viral vaccines.

INTRODUCTION

This application claims benefit of U.S. Provisional Application No.60/022,621, filed Aug. 22, 1995.

1. Technical Field

The present invention relates to methods for the production of virus forvaccine production in cell culture.

2. Background

Effective control of influenza pandemics depends on early vaccinationwith the inactivated virus produced from newly identified influenzastrains. However, for more effective pandemic control, improvements inthe manufacturing and testing of the vaccine are needed. Influenzaviruses undergo very frequent mutations of the surface antigens.Consequently, vaccine manufacturers cannot stock-pile millions of dosesfor epidemic use. Current influenza control methods demand constantinternational surveillance and identification of any newly emergentstrains coupled with vaccine production specific for the newlyidentified strains. Current influenza vaccine production, which requiresthe use of embryonated eggs for virus inoculation and incubation, iscumbersome and expensive. It can also be limited by seasonalfluctuations in the supply of suitable quality eggs. Thus, forproduction of mass doses of monovalent vaccine in a short time, it wouldbe advantageous to develop alternate, egg-independent productiontechnology. In this respect, production of an influenza vaccine on astable cell line may solve many of the problems in mass production.However, the yield of human influenza viruses on tissue culture isdisappointingly much lower than in embryonated eggs (Tannock et al.Vaccine 1985 3:333-339). To overcome these limitations and improve thequality of vaccines, it would be advantageous to develop cell culturelines which provide an enhanced yield of virus over those currentlyavailable.

In using mammalian cell lines for whole virion vaccine production, acommon problem for vaccine manufacturers is that mammalian cells haveintrinsic antiviral properties, specifically, the interferon (IFN)system, which interferes with viral replication. IFNs can be classifiedinto two major groups based on their primary sequence. Type Iinterferons, IFN-α and IFN-β, are encoded by a super family ofintronless genes consisting of the IFN-α gene family and a single IFN-βgene. Type II interferon, or IFN-γ, consists of only a single type andis restricted to lymphocytes (T-cells and natural killer cells). Type Iinterferons mediate diverse biological processes including induction ofantiviral activities, regulation of cellular growth and differentiation,and modulation of immune functions (Sen, G. C. & Lengyel, P. (1992) J.Biol. Chem. 267, 5017-5020; Pestka, S. & Langer, J. A. (1987) Ann. Rev.Biochem. 56, 727-777). The induced expression of Type I IFNs, whichinclude the IFN-α and IFN-β gene families, is detected typicallyfollowing viral infections. Many studies have identified promoterelements and transcription factors involved in regulating the expressionof Type I IFNs (Du, W., Thanos, D. & Maniatis, T. (1993) Cell 74,887-898; Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami,T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham,A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki, H., Ohashi, P.S., Paige, C. J., Taniguchi, T. & Mak, T. W. (1993) Cell 75, 83-97;Tanaka, N. & Taniguchi, T. (1992) Adv. Immunol. 52, 263-81). However, itremains unclear what are the particular biochemical cues that signifyviral infections to the cell and the signaling mechanisms involved (fora recent review of the interferon system see Jaramillo et al. CancerInvestigation 1995 13:327-337).

IFNs belong to a class of negative growth factors having the ability toinhibit growth of a wide variety of cells with both normal andtransformed phenotypes. IFN therapy has been shown to be beneficial inthe treatment of human malignancies such as Kaposi's sarcoma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma and hairy cell leukemia aswell as the treatment of infectious diseases such as papilloma virus(genital warts) and hepatitis B and C (reviewed by Gutterman Proc. NatlAcad Sci. 91:1198-1205 1994). Recently, genetically-engineeredbacterially-produced IFN-β was approved for treatment of multiplesclerosis, a relatively common neurological disease affecting at least250,000 patients in the US alone.

IFNs elicit their biological activities by binding to their cognatereceptors followed by signal transduction leading to induction ofIFN-stimulated genes (ISG). Several of them have been characterized andtheir biological activities examined. The best studied examples of ISGsinclude a double-stranded RNA (dsRNA) dependent kinase (PKR, formerlyknown as p68 kinase), 2'-5'-linked oligoadenylate (2-5A) synthetase, andMx proteins (Taylor J L, Grossberg S E. Virus Research 1990 15:1-26.;Williams BRG. Eur. J. Biochem. 1991 200:1-11). Human Mx A protein is a76 kD protein that inhibits multiplication of influenza virus andvesicular stomatitis virus (Pavlovic et al. (1990) J. Viol. 64,3370-3375).

2'-5' Oligoadenylate synthetase (2-5A synthetase) uses ATP to synthesizeshort oligomers of up to 12 adenylate residues linked by2'-5'-phosphodiester bonds. The resulting oligoadenylate moleculesallosterically activate a latent ribonuclease, RNase L, that degradesviral and cellular RNAs. The 2-5A synthetase pathway appears to beimportant for the reduced synthesis of viral proteins in cell-freeprotein-synthesizing systems isolated from IFN-treated cells andpresumably for resistance to viral infection in vivo at least for someclasses of virus.

PKR (short for protein kinase RNA-dependent) is the only identifieddouble-stranded RNA (dsRNA)-binding protein known to possess a kinaseactivity. PKR is a serine/threonine kinase whose enzymatic activationrequires binding to dsRNA or to single-stranded RNA presenting internaldsRNA structures and consequent autophosphorylation (Galabru, J. &Hovanessian, A. (1987) J. Biol. Chem. 262, 15538-15544; Meurs, E.,Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. G. &Hovanessian, A. G. (1990) Cell 62, 379-390). PKR has also been referredto in the literature as dsRNA-activated protein kinase, P1/e1F2 kinase,DAI or dsI for dsRNA-activated inhibitor, and p68 (human) or p65(murine) kinase. Analogous enzymes have been described in rabbitreticulocytes, different murine tissues, and human peripheral bloodmononuclear cells (Farrel et al. (1977) Cell 11, 187-200; Levin et al.(1978) Proc. Natl Acad. Sci. USA 75, 1121-1125; Hovanessian (1980)Biochimie 62, 775-778; Krust et al. (1982) Virology 120, 240-246;Buffet-Janvresse et al. (1986) J. Interferon Res. 6, 85-96). The bestcharacterized in vivo substrate of PKR is the alpha subunit ofeukaryotic initiation factor-2 (eIF-2a) which, once phosphorylated,leads ultimately to inhibition of cellular and viral protein synthesis(Hershey, J. W. B. (1991) Ann. Rev. Biochem. 60, 717-755). PKR canphosphorylate initiation factor e1F-2α in vitro when activated bydouble-stranded RNA (Chong et al. (1992) EMBO J. 11, 1553-1562). Thisparticular function of PKR has been suggested as one of the mechanismsresponsible for mediating the antiviral and antiproliferative activitiesof IFN-α and IFN-β. An additional biological function for PKR is itsputative role as a signal transducer. Kumar et al. demonstrated that PKRcan phosphorylate IκBα, resulting in the release and activation ofnuclear factor κB (NF-κB) (Kumar, A., Haque, J., Lacoste, J., Hiscott,J. & Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. USA 91,6288-6292). Given the well-characterized NF-κB site in the IFN-βpromoter, this may represent a mechanism through which PKR mediatesdsRNA activation of IFN-β transcription (Visvanathan, K. V. &Goodbourne, S. (1989) EMBO J. 8, 1129-1138).

The catalytic kinase subdomain of PKR (i.e., of p68 (human) kinase andp65 (murine) kinase) has strong sequence identity (38%) with the yeastGCN2 kinase (Chong et al. (1992) EMBO J. 11, 1553-1562; Feng et al.(1992) Proc. Natl. Acad. Sci. USA 89, 5447-5451). Recombinant p68 kinaseexpressed in yeast Saccharomyces cerevisiae exhibits agrowth-suppressive phenotype. This is thought to be attributed to theactivation of the p68 kinase and subsequent phosphorylation of the yeastequivalent of mammalian e1F2α (Chong et al.; Cigan et al. (1982) Proc.Natl. Acad. Sci. USA 86, 2784-2788).

The present inventor have surprisingly discovered by manipulating theexpression of certain ISGs that manipulation of ISGs can have beneficialuses. They have discovered that suppression of the expression of the PKRprotein or the 2-5A synthetase protein or both results in asubstantially higher viral yield from virus-infected cells which isuseful for enhancing the production of vaccines in animal cell culture.

Relevant Literature

A common approach to examine the biological role of PKR involves thegeneration of mutants deficient in the kinase activities. Since PKRpossesses a regulatory site for dsRNA binding and a catalytic site forkinase activity, investigators have used block deletion or site-directedmutagenesis to generate mutants at the regulatory or catalytic site. APKR dominant negative mutant, Arg²⁹⁶ !PKR, which contains a single aminoacid substitution of arginine for the invariant lysine in the catalyticdomain II at position 296 has been described (Visvanathan, K. V. &Goodboume, S. (1989) EMBO J. 8, 1129-1138; D'Addario, M., Roulston, A.,Wainberg, M. A. & Hiscott, J. (1990) J. Virol. 64, 6080-6089). Thismutant protein Arg²⁹⁶ !PKR can specifically suppress the activity ofendogenous wild-type PKR in vivo. Additional mutants have been generatedby altering the dsRNA binding motifs. For example, Feng et al. (ProcNatl Acad Sci USA 1992 89:5447-5451) abolished dsRNA binding ability ofPKR by deletional analysis to obtain mutants with deletions betweenamino acid residues 39-50 or 58-69. Similarly, other investigators havemutated amino acid residues in the N-terminal region to suppress dsRNAbinding ability leading to loss of PKR enzymatic activities (Green S R,Mathews M B. Genes & Development 1992 6:2478-2490; McCormack S J, OrtegaL G, Doohan J P, Samuels C E. Virology 1994 198:92-99). A recent articlehas further identified two amino acid residues that are absolutelyrequired for dsRNA binding, namely glycine 57 and lysine 60 (McMillan NA J, Carpick B W, Hollis B, Toone W M, Zamanian-Daryoush, and Williams BR G. J. Biol. Chem. 1995 270:2601-2606). Mutants in these positions wereshown to be unable to bind dsRNA in vitro and possessed noantiproliferative activity in vivo when expressed in murine macrophagecells.

The physiological significance of the loss of PKR activity in vivo hasbeen examined in animals. Catalytically inactive PKR mutants (includingArg²⁹⁶ !PKR) when transfected into NIH 3T3 (mouse fibroblast) cellscaused suppression of endogenous PKR activity in the transfectants. Whenadministered to nude mice, these transfected cells caused tumorformation suggesting a tumor suppressor activity for PKR (Koromilas, A.E., Roy, S., Barber, G. N., Katze, M. G. & Sonenberg, N. (1992) Science257, 1685-1689; Meurs, E. F., Galabru, J., Barber, G. N., Katze, M. G. &Hovanessian, A. G. (1993) Proc. Natl. Acad. Sci. USA 90, 232-236). Meurset al. (J. Virol. 1992 66:5805) produced stable transfectants of NIH 3T3cells with either a wild type (wt) PKR gene or a dominant negativemutant under control of a CMV promoter and showed that onlytransfectants receiving the wt clone were partially resistant toinfection with encephalomyocarditis virus (EMCV). Lee et al. (Virol.1993 193:1037) constructed a recombinant vaccinia virus vectorcontaining the PKR gene under control of an inducible promoter andshowed that in HeLa cells infected with the recombinant virus andinduced resulted in an inhibition of the vaccinia virus protein and anoverall decrease in viral yield. Henry et al. (J. Biol. Regulators andHomeostatic Agents 1994 8:15) showed that reoviral mRNAs containing aPKR activator sequence are poorly expressed in comparison with otherreoviral mRNAs but that addition of 2-aminopurine, a PKR inhibitor, ortransfection with a dominant negative PKR mutant, specifically increasedthe expression of mRNA containing the activator sequence. Maran et al.(Science 1994 265:789) showed that HeLa cells that were selectivelydeleted for PKR mRNA by treatment with PKR antisense oligos linked to2'-5' oligoA were unresponsive to activation of nuclear factor-κB by thedsRNA poly(I):poly(C).

Several strategies have been utilized in the effort to improve the yieldof virus obtained from cell culture for vaccine production. Differentcell types have been tested to obtain the best cell line for optimumgrowth of specific viruses. The diploid human embryonic lung cell lines,MRC-5 and WI-38, have been developed specifically for vaccine production(see Pearson Devel. Biol. Standard. 1992 76:13-17; MacDonald, C.Critical Reviews Biotech. 1990 10:155-178; Wood et al. Biologicals 199018:143-146). Other attempts to improve vaccine production from cellculture include use of a low protein serum replacement factor (Candal etal. Biologicals 1991 19:213-218), and treatment of the cell culture withproteolytic enzymes (U.S. Pat. No. RE 33,164).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forenhanced vaccine production in cell culture. It is another object of theinvention to provide methods for the evaluation of antiviral compoundsand for the identification and culture of viral pathogens.

These object are generally accomplished by providing animal cellcultures in which the expression of the interferon genes issubstantially decreased from the normal level of expression. This may beeffected by manipulating the level of expression of factors thatfunction in vivo to regulate the interferon level, including interferontranscriptional regulators (for example, IRF1), interferon receptors andinterferon stimulated gene products (for example PKR and 2-5Asynthetase).

These objects are particularly accomplished by providing various methodsusing animal cell cultures in which the level of interferon-mediatedantiviral protein activity, particularly for double-stranded RNAdependent kinase (PKR) and 2'-5' Oligoadenylate synthetase (2-5Asynthetase), is significantly decreased from the normal levels. Amongthe various methods provided are methods for vaccine production methods,for determining the antiviral activity of a compound, and methods fordetecting a virus in a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. PKR activity and protein levels in U937-derived stabletransfectant cell lines. (A) Functional PKR activity was determinedusing a poly(I):poly(C)-cellulose assay for PKR autophosphorylation.Cell extracts were prepared from the different U937 transfectant celllines following incubation with (+) or without (-) recombinant humanIFN-α2 (200 U/mL) as indicated, while L929 cells were similarly treatedwith mouse IFN-α/β. Lane 1, HeLa; lanes 2 and 3, U937-neo; lane 4,U937-AS1; lane 5, U937-AS3; lane 6, U937-M13; lane 7, U937-M22; lane 8,L929. Positions of the human (68 kDa) and mouse (65 kDa) PKR proteins,and the molecular size standards (80 and 50 kDa) are indicated. (B) Cellextracts were prepared as above after induction with IFN-α or -γ and PKRprotein levels were determined by Western blot analysis.

FIGS. 2A and 2B. Kinetics of EMCV replication are enhanced inPKR-deficient cells. The different U937 cell lines were challenged withEMCV at 0.1 (A) or 0.001 (B) TCID₅₀ /cell. Samples were harvested at theindicated times and viral yields were measured in terms of TCID₅₀.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relies upon the discovery by the inventor that thelevel of interferon production in cells can be regulated by manipulatingthe expression or activity of certain factors that normally regulateinterferon expression and activity in vivo. These factors includecertain interferon-specific transcriptional regulators, particularlyIRF1, certain interferon receptors, as well as the gene products ofcertain interferon simulated genes (also called interferon-mediatedantiviral responses), particularly PKR and 2-5A synthetase. Suppressionor elimination of the expression or activity of any of these factorswill result in a lower than normal level of expression of interferongenes. One consequence of this lower than normal interferon expressionlevel is an increased permissiveness of the cell to viral replication.An increased permissiveness of the cell to viral reproduction means thatgreater viral production can be achieved in that cell relative to a cellhaving normal interferon expression. Cells having an increasedpermissiveness to viral replication are useful for a number ofapplications including vaccine production, sensitive detection of lowlevels of virus and for the evaluation of antiviral compounds.

The present inventor has surprisingly found that animal cells that aredeficient in interferon-mediated antiviral responses, particularly cellsdeficient in dsRNA dependent kinase, 2'-5' Oligoadenylate synthetase orboth, produce a higher viral yield when infected with an animal virusthan cells with normal levels of these proteins. Increases of viralyield by as much as 10³ to 10⁴ or more can be obtained using the methodof the present invention. The ability to obtain high yields of virus inPKR- or 2-5A synthetase-deficient cell culture makes it possible toproduce large amounts of virus within a short time. This is particularlyimportant for production of viral vaccines, most particularly for RNAvirus, including influenza virus. The increased permissiveness of thedeficient cells to viral replication makes them useful in a method forevaluating antiviral drugs in cell culture and in a method for detectingviral pathogens.

On aspect of the present invention provides a method for production of aviral vaccine in cell culture which comprises (a) infecting a cellculture with a donor strain animal virus, wherein said cell culture isdeficient in the activity of the gene product of aninterferon-stimulated gene, (b) culturing the infected cell cultureunder conditions sufficient to provide efficient virus growth, and (c)harvesting the virus produced. The harvested virus may be additionallyprepared for vaccine use by purification, for instance by sterilefiltration, ultrafiltration and/or concentration by columnchromatography or other methods. The harvested virus may optionally betreated to inactivate the virus for the production of killed viralvaccines.

In a preferred embodiment, the cell culture is deficient in PKRactivity. By PKR-deficient is meant that the PKR activity is less than5% of the normal level of PKR activity. By normal level of PKR activityis meant the PKR activity observed in the parental cell culture fromwhich the stable PKR-deficient cells are obtained or, if thePKR-deficiency is transiently induced, the PKR activity level observedin the cells before induction to PKR-deficiency. Preferably, thePKR-deficient cells have less than 1% of the normal level of PKRactivity, more preferably the PKR-deficient cells have less than 0.1% ofthe normal level of PKR activity. By PKR activity is meant the abilityto mediate the antiviral and antiproliferative activities of IFN-α andIFN-β, the ability to phosphorylate initiation factor e1F-2α, or theability to phosphorylate IκBα to release nuclear factor κB. By PKR ismeant human p68 kinase or any analog or homolog of human p68 kinase. Byanalog of human p68 kinase is meant any double-stranded RNA-dependentkinase that mediates ds-RNA activation of interferon transcription.Typically, such ds-RNA dependent kinases are p68 kinase equivalentspresent in other species, such as, for example, rabbits or mice and indifferent tissues among the various species. For example, murine p65kinase is an analog of human p68 kinase. Another example of an analog ofp68 kinase has been described in human peripheral blood mononuclearcells (Farrel et al.) By homolog is meant a protein homologous to atleast one domain of human p68 kinase, such as, for example, thedsRNA-binding domain or the kinase domain. One such functional kinasehomolog is yeast GCN2 kinase.

PKR-deficient cells can be obtained by any of a variety of methods thatare well-known in the art. PKR-deficient mutants can be stablyPKR-deficient or may be transiently induced to PKR-deficiency.Techniques for producing stable PKR-deficient mutants include, but arenot limited to, random or site-directed mutagenesis (for example, Deng WP, and Nickoloff J A Analytical Biochemistry 1992 200:81-88; Busby S,Irani M, Crombrugghe B. J. Mol Biol 1982 154:197-209), targeted genedeletion ("gene knock-out") (for example, Camper S A, et al. Biology ofReproduction 1995 52:246-257; Aguzzi A, Brandner S, Sure U et al. BrainPathology 1994 4:3-20), transfection with PKR antisense polynucleotides(for example, Lee et al. Virology 1993 192:380-385) and transfectionwith a PKR dominant negative mutant gene.

A PKR dominant mutant is a PKR mutant for which only a single alleleneed be expressed in order to suppress normal PKR activity. PKR dominantmutant genes include a mutant human p68 kinase, a mutant murine p65kinase, and mutants of any other ds-RNA dependent kinases or mutants ofanalogs or homologs of human p68 kinase that suppress normal PKRactivity, for example Arg²⁹⁶ !PKR (Meurs et al. J. Virol. 199266:5805-5814). Examples of other PKR dominant mutants include mutants ofPKR obtained from rabbit reticulocytes, different mouse tissues andhuman peripheral blood mononuclear cells (Farrel et al., Levin et al.,Hovanessian, Krust et al., Buffet-Janvresse et al.) PKR dominant mutantsinclude mutants of functional homologs that suppress protein synthesisby interfering with initiation factor phosphorylation, particularlyphosphorylation of e1F-2α. One such functional kinase homolog mutant isa mutant of yeast GCN2 kinase.

Techniques for producing cells that are transiently PKR-deficientinclude, but are not limited to, use of 2'-5'oligoadenylate-linked PKRantisense oligonucleotides (Maran, A., Maitra, R. K., Kumar, A., Dong,B., Xiao, W., Li, G., Williams, B. R. G., Torrence, P. F. & Silverman,R. H. (1994) Science 265, 789-792) or specific inhibitors of the PKRprotein, such as 2-aminopurine (Marcus, P. I. & Sekellick, M. J. (1988)J. Gen. Virol. 69, 1637-45, Zinn, K., Keller, A., Whittemore, L. A. &Maniatis, T. (1988) Science 240, 210-3) as well as other competitiveinhibitors that can block phosphorylation of PKR substrates, orinhibitors that can block double-stranded RNA binding. TransientlyPKR-deficient cell cultures can be obtained by culturing a cell line inthe presence of such antisense oligonucleotides or inhibitors.

Preferably for use in the method of the present invention, cell cultureswill be stably PKR-deficient. Typically, PKR-deficient cell cultures areproduced by transfection of a parent cell line, preferably a cell linecurrently used in vaccine production, preferably MRC-5, WI-38, or Vero(African Green Monkey cell), with a vector containing a functional PKRantisense gene construct or a PKR dominant negative mutant constructfollowed by selection of those cells that have received the vector. Afunctional PKR antisense gene construct may be prepared by conventionalmethods; for example, by cloning a PKR cDNA such as that described inMeurs et al. (Cell 1990 62:379-390), in an antisense orientation, underthe control of an appropriate promoter, for example a CMV promoter. APKR dominant negative mutant construct can be prepared by cloning thecDNA for a PKR dominant negative mutant, for example the cDNA for Arg²⁹⁶!PKR, under the control of an appropriate promoter.

Preferably the PKR mutant gene constructs are cloned under the controlof an inducible promoter to reduce the risk of tumor formation by thesePKR-deficient cells since the cells are to be used for vaccineproduction in the methods of the invention. This method will ensure thesafety of the vaccines produced by these cells. The loss of PKR activityhas been associated with tumor formation (Koromilas et al.; Meurs etal.). Although the harvested virus can be purified from cell culturecomponents, there nevertheless remains a risk that some PKR-deficientcells would be carried over into the final vaccine preparation. If PKRactivity remains constitutively suppressed, these cells may potentiallybecome tumorigenic. This would create potential health risk for thevaccine recipient. However, if an inducible promoter is used to controlexpression of the gene construct, endogenous PKR activity would berestored upon removal of the inducer. Suitable inducible promotersinclude a lac promoter, a heat shock promoter, a metallothioneinpromoter, a glucocorticoid promoter, or any other inducible promoterknown to one skilled in the art.

Other ways of constructing similar vectors, for example using chemicallyor enzymatically synthesized DNA, fragments of the PKR cDNA or PKR gene,will be readily apparent to those skilled in the art. Transfection ofthe parental cell culture is carried out by standard methods, forexample, the DEAE-dextran method (McCutchen and Pagano, 1968, J. Natl.Cancer Inst. 41:351-357), the calcium phosphate procedure (Graham etal., 1973, J. Virol. 33:739-748) or by any other method known in theart, including but not limited to microinjection, lipofection, andelectroporation. Such methods are generally described in Sambrook etal., Molecular Cloning: A laboratory manual, 2nd Edition, 1989, ColdSpring Harbor Laboratory Press. Transfectants having deficient PKRactivity are selected. For ease of selection, a marker gene such asneomycin phosphotransferase II, ampicillin resistance or G418resistance, may be included in the vector carrying the antisense ormutant gene. When a marker gene is included, the transfectant may beselected for expression of the marker gene (e.g. antibiotic resistance),cultured and then assayed for PKR activity.

Residual PKR activity in PKR-deficient cells can be determined by any ofa number of techniques that are well-known in the art. The activity ofPKR can be determined directly by, for example, an autophosphorylationassay such as that described in Maran et al. (Science 265:789-792 1994)or Silverman et al. (Silverman, R. H., and Krause, D. (1986) inInterferons: A practical approach. Morris, A. G. and Clemens, M. J.,eds. pp. 71-74 IRL Press, Oxford-Washington, DC.). Typically, anautophosphorylation assay for PKR activity is carried out as follows.Extracts from cells to be examined for PKR activity which containapproximately 100 μg of protein are incubated with 20 μl ofpoly(I):poly(C)-cellulose beads for 60 min on ice. The kinase isimmobilized and activated on the beads. After washings of thepolynucleotide cellulose-bound kinase fractions, an autophosphorylationreaction is performed at 30° C. for 30 min in an assay solution. Theassay solution contains 1 μCi of γ³² P!ATP, 1.5 mM magnesium acetate, 10μM ATP pH 7.5, 0.5% NP 40, and 100 μg/ml leupeptin. The samples areheated at 90° C. for 3 min in gel sample buffer containing sodiumdodecyl sulfate (SDS) and the proteins are analyzed by 10%SDS-polyacrylamide gel electrophoresis. The gels are dried andautoradiographs are prepared using XAR-5 X-ray film (KodaK).

Residual PKR activity may also be determined indirectly by assaying forthe presence of the PKR protein, for example by Western blot with PKRspecific antibodies, or for the presence of PKR RNA, for example byNorthern blot with oligonucleotide or cDNA probes specific for PKR. Aswill be readily apparent, the type of assay appropriate fordetermination of residual PKR activity will in most cases depend on themethod used to obtain the PKR-deficient phenotype. If, for example, themethod used to produce the PKR-deficient cell results in suppression orelimination of PKR gene expression (for example, gene knock-out),analysis techniques that detect the presence of mRNA or cDNA (e.g.Northern or Southern blots) or the presence of the protein (e.g. Westernblot) or that detect the protein activity may be useful to determine theresidual PKR activity in the PKR-deficient cells. On the other hand, ifthe method used to produce the PKR-deficient cells results in inhibitionof the protein rather than elimination of expression of the gene (forinstance, transfection with a vector carrying a dominant negative PKRmutant), an autophosphorylation assay is more appropriate than a Westernblot for determination of the residual PKR activity.

In another embodiment, the present invention provides a method forproduction of a viral vaccine in a cell culture that is deficient in2'-5' Oligoadenylate synthetase activity. A cell culture deficient in2-5A synthetase can be isolated in a similar fashion to cell culturesdeficient in PKR, for example, random or site-directed mutagenesis,targeted gene deletion of the 2-5A synthetase genes or transfection withantisense 2-5A synthetase constructs. By 2-5A synthetase-deficient ismeant that the 2-5 A synthetase activity is less than 5% of the normallevel of 2-5A synthetase activity. By normal level of 2-5A synthetaseactivity is meant the 2-5A synthetase activity observed in the parentalcell culture from which the stable 2-5A synthetase-deficient cells areobtained or, if the 2-5A synthetase-deficiency is transiently induced,the 2-5A synthetase activity level observed in the cells beforeinduction to 2-5A synthetase-deficiency. Preferably, the 2-5Asynthetase-deficient cells have less than 1% of the normal level of 2-5Asynthetase activity, more preferably the 2-5A synthetase-deficient cellshave less than 0.1% of the normal level of 2-5A synthetase activity.Residual 2-5A synthetase activity in 2-5A synthetase-deficient cells canbe determined by methods similar to those used for determining residualPKR activity, that is, Western blots using 2-5A synthetase specificantibodies, Northern blots using oligonucleotide or cDNA probes specificfor 2-5A synthetase or enzyme activity assays (see, Read et al. J.Infect. Dis. 1985 152:466-472; Hassel and Ts'o J. Virol. Methods 199450:323-334). Typically, 2-5A synthetase activity is determined asfollows. Cells to be assayed are treated with IFN-α₂ (100 U/1 ml in RPMIplus 10% fetal bovine serum). Briefly, the cell cultures are incubatedfor 18 hr at 37° C., washed and the cell pellets are treated with celllysis buffer for 10 min at 4° C. Aliquots of the cellular extract areincubated with poly(I):poly(C)-agarose beads for 30 min at 30° C., toallow for binding as well as activation of the 2-5A synthetase enzyme.The beads are washed and then incubated in an assay solution containing3 mM ATP, 4 μCi ³ H-ATP per assay sample, and 20 mM Hepes buffer pH 7.5for 20 hr at 30° C. Following incubation, the samples are heated at 90°C. to inactivate the enzyme, followed by treatment with bacterialalkaline phosphatase (BAP). The 2-5 oligoA synthesized is resistant toBAP. The amount of 2-5 oligo A is determined by spotting a sample ontofilter paper, washing and counting the ³ H radioactivity using ascintillation counter. The amount of oligoA product produced iscorrelated with the enzyme activity by conventional methods.Alternatively, 2-5A synthetase can be assayed by a radioimmune andradiobinding method (Knight M, et al. Radioimmune, radiobinding and HPLCanalysis of 2-5A and related oligonucleotides from intact cells Nature1980 288:189-192).

It will be apparent that cell cultures deficient in both PKR activityand 2-5A synthetase activity can be made by a combination of the methodsdescribed above. The doubly deficient cell cultures can be preparedeither sequentially (that is, by first selecting cultures deficient inone activity and then using that cell culture as the starting materialfor preparing the second deficient culture) or simultaneously (selectionfor both deficiencies at once).

In another embodiment, the present invention provides a method forproduction of a viral vaccine in a cell culture that is deficient inhuman M×A protein activity. A cell culture deficient in human M×Aprotein activity can be isolated in a similar fashion to cell culturesdeficient in PKR, for example, random or site-directed mutagenesis,targeted gene deletion of the M×A genes or transfection with antisenseM×A constructs. By M×A protein-deficient is meant that the M×A activityis less than 5% of the normal level of M×A activity. By normal level ofM×A activity is meant the M×A activity observed in the parental cellculture from which the stable M×A-deficient cells are obtained or, ifthe M×A-deficiency is transiently induced, the M×A activity levelobserved in the cells before induction to M×A-deficiency. Preferably,the M×A-deficient cells have less than 1% of the normal level of M×Aactivity, more preferably the M×A-deficient cells have less than 0.1% ofthe normal level of M×A activity. Residual M×A activity in M×A-deficientcells can be determined by methods similar to those used for determiningresidual PKR activity, that is, Western blots using M×A specificantibodies, Northern blots using oligonucleotide or cDNA probes specificfor M×A or enzyme activity assays (Garber et al. (1991) Virology 180,754-762; Zurcher et al. (1992) Journal of Virology 66, 5059-5066).Typically, M×A activity is determined as described in Zurcher et al.

In yet another embodiment, the present invention provides a method forproduction of a viral vaccine in a cell culture that is deficient ininterferon responsiveness. By interferon responsiveness is meant theability of a cell to respond to stimulation by interferon. A cellculture deficient in interferon responsiveness can be obtained byculturing the cells in the presence of an inhibitor of an interferonreceptor. Alternatively, cells can be engineered to express, in theabsence of a normal interferon receptor, a mutant interferon receptorthat is unresponsive to interferon.

In another embodiment, the present invention provides a method forproduction of a viral vaccine in a cell culture that is deficient ininterferon-specific transcriptional regulators. One suchinterferon-specific transcriptional regulator is IRF1. Cells stablydeficient in interferon-specific transcriptional regulators can beobtained by any of a number of techniques well known in the art, suchas, for example, random or site-directed mutagenesis, targeted genedeletion, or transfection with antisense vectors. Transiently deficientcells can be obtained by culturing cells in the presence of antisenseoligonucleotides or specific inhibitors of interferon transcription.

The method of the present invention can be practiced with a variety ofanimal cell cultures, including primary cell cultures, diploid cellcultures and continuous cell cultures. Particularly useful are cellcultures that are currently used for the production of vaccine, mostparticularly those cell cultures that have been approved for vaccineproduction by the USFDA and or WHO, for example, MRC-5, a human diploidcell line from fetal lung tissue (Nature Lond. 1970 227:168-170) andWI-38, a human diploid cell line derived from embryonic lung tissue (Am.J. Hyg. 1962 75:240; First International Conference on Vaccines AgainstViral and Rickettsial Diseases of Man, Pan American Health Organization,Pub. No. 147: 581 May 1981). Also useful are Chang liver cells (Chang, RS Proc. Soc. Exp. Biol. Med. 1954 87:440), U937 human promonocytic cells(Sundstrom et al. Int. J. Cancer 1976 17:565-577), Vero cells, MRC-9cells, 1MR-90 cells, 1MR-91 cells and Lederle 130 cells (Biologicals18:143-146 1991). U937 cells are particularly useful for viruses thatinfect immune cells expressing CD4, for example, HIV. For a generalreview of cell cultures used in the production of vaccines see Grachev,V.P. in Viral Vaccines Mizrahi, A. ed. pages 37-67 1990 Wiley-Liss. Theparticular cell culture chosen will depend on the virus which is to beproduced; in general, the cell culture will be derived from the specieswhich is the natural host for the virus, although this is not essentialfor the practice of the present invention (for example, human virus canbe grown on a canine kidney cell line (MDCK cells) or a green monkeykidney cell line (Vero cells; Swanson et al. J. Biol. Stand. 198816:311)). Typically, the cells chosen will be PKR-deficient or 2-5Asynthetase-deficient derivatives of cells or cell lines known to be anappropriate host for the virus to be produced. For example, forinfluenza virus and hepatitis A virus vaccines, preferred host cells arederivatives of MRC-5. For HIV vaccine production, preferred host cellsare derivatives of U937, H9, CEM or CD4-expressing HUT78 cells. Celllines used for the production of vaccines are well known and readilyavailable from commercial suppliers, for example, American Type CultureCollection.

The infection of the interferon-mediated antiviral response-deficientcells with donor virus according to the present invention is carried outby conventional techniques (see for example Peetermans, J. Vaccine 199210 supp 1:S99-101; Shevitz et al. in Viral Vaccines Mizrahi, a. ed. pp1-35 1990 Wiley-Liss). Typically, virus is added to the cell culture atbetween 0.001 to 0.5 TCID₅₀ per cell, preferably at 0.01 to 0.10 TCID₅₀per cell, but will vary as appropriate for the particular virus and cellhost being used. As is readily apparent to one of ordinary skill in theart, every cell of the cell culture need not be infected initially forefficient viral production. The infected cells are cultured underconditions appropriate for the particular cells and viral production atvarious times after infection is monitored. Viral production can bemonitored by any of a number of standard techniques includingplaque-forming unit assays, TCID₅₀ assays or hemagglutination inhibitionassays (Robertson et al. J. Gen. Virol. 1991 72:2671-2677). The infectedcells are cultured under conditions sufficient to provide efficientviral growth. The cells can be cultured until maximum viral productionis achieved as indicated by a plateauing of the viral yield. The virusis harvested by standard techniques and substantially purified fromother cellular components (see for example, Peetermans 1992). Theharvested virus may be used as a live viral vaccine, either fullyvirulent or attenuated, or may be inactivated before use by methods thatare well-known in the art, for example, by treatment with formaldehyde(Peetermans, J Vaccine 1992 10 Suppl 1:S99-101; U.S. Pat. No. RE33,164).

The vaccine may be available in dry form, to be mixed with a diluent, ormay be in liquid form, preferably in aqueous solution, eitherconcentrated or ready to use. The vaccine is administered alone or incombination with pharmaceutically acceptable carriers, adjuvants,preservatives, diluents and other additives useful to enhanceimmunogenicity or aid in administration or storage as are well-known inthe art. Suitable adjuvants include aluminum hydroxide, alum, aluminumphosphate, Freunds or those described in U.S. Pat. Nos. 3,790,665 and3,919,411. Other suitable additives include sucrose, dextrose, lactose,and other non-toxic substances. The vaccines are administered to animalsby various routes, including intramuscular, intravenous, subcutaneous,intratracheal, intranasal, or by aerosol spray and the vaccines arecontemplated for the beneficial use in a variety of animals includinghuman, equine, avian, feline, canine and bovine.

The method of the present invention can be practiced with a variety ofdonor animal viruses. By donor virus is meant the particular viralstrain that is replicated in vitro to produce the vaccine. Theparticular donor animal virus used will depend upon the viral vaccinedesired. Donor viruses currently used for vaccine production arewell-known in the art and the method of the present invention can bereadily adapted to any newly identified donor virus. Preferred donorviruses include human influenza virus, especially influenza A (H3N2) andinfluenza A (H1N1) (see U.S. Pat. No. 4,552,758; ATCC Nos. VR-2072,VR-2073, VR-897); influenza A described in U.S. Pat. No. 3,953,592;influenza B (U.S. Pat. No. 3,962,423; ATCC Nos. VR-786, VR-791); andParainfluenza 1 (Sendai virus) (Cantell et al. Meth. Enzymol.78A:299-301 1980; ATCC No.VR-907). The donor virus can be identical tothe viral pathogen or may be a naturally-occurring attenuated form, anattenuated form produced by serial passage through cell culture or arecombinant or reassortant form. Any viral strain may be used as donorvirus provided that it retains the requisite antigenicity to affordprotection against the viral pathogen. The method of the presentinvention is particularly useful with attenuated or poorly replicatingdonor viruses.

Some of the vaccines that can be provided by the methods of the presentinvention include, but are not limited to, human vaccines forpoliovirus, measles, mumps, rubella, hepatitis A, influenza,parainfluenza, Japanese encephalitis, cytomegalovirus, HIV, Dengue fevervirus, rabies and Varicella-zoster virus, as well as many non-humananimal vaccines including, for example, vaccines for feline leukemiavirus, bovine rhinotracheitis virus (red nose virus), cowpox virus,canine hepatitis virus, canine distemper virus, equine rhinovirus,equine influenza virus, equine pneumonia virus, equine infectious anemiavirus, equine encephalitis virus, ovine encephalitis virus, ovine bluetongue virus, rabies virus, swine influenza virus and simianimmunodeficiency virus. As will be apparent from the foregoing, themethod of the present invention is not limited to vaccine production forhuman viruses but is equally suitable for production of non-human animalviral vaccines.

Another aspect of the present invention provides a method for evaluatingthe activity of antiviral compounds. Due to the increased permissivenessof the PKR-deficient cells to viral replication, the cells are useful ina sensitive assay for assessing the effectiveness of antiviralcompounds. In this aspect, the present invention comprises the steps of(a) treating a virus, virus-infected host cells or host cells prior tovirus infection with the antiviral compound and (b) assaying for thepresence of remaining infectious virus by exposure under infectiveconditions of a PKR-deficient or 2-5A synthetase-deficient indicatorcell culture.

In this aspect, the virus against which the antiviral compound is to betested may be treated directly with the compound. In this case, thetreated virus may then be analyzed directly for the presence ofremaining infectious virus by exposure under infective conditions of aPKR-deficient or 2-5A synthetase-deficient indicator cell culture to analiquot of the treated virus, culturing for a time sufficient to allowreplication of any remaining infectious virus and analyzing theindicator culture for the presence of the replicated virus.Alternatively, the virus against which the antiviral compound is to betested may be used to infect a host cell culture, the infected host cellculture is then treated with the antiviral compound. A cell extract ofthe treated infected host cell culture is prepared by conventionaltechniques and an aliquot of the extract is analyzed for the presence ofremaining infectious virus by exposure to a PKR-deficient or 2-5Asynthetase-deficient indicator cell culture as described above. Inanother alternative, the host cell culture may be treated with theantiviral compound prior to infection with the virus rather than afterinfection. The treated cells are then infected with the virus againstwhich the antiviral compound is to be tested, cultured and analyzed forthe presence of replicated virus. The particular treatment regime chosenwill depend upon the known or postulated mode of action of the antiviralcompound and will be readily within the determination of one skilled inthe art. By exposure under infective conditions is intended the bringingtogether the deficient indicator cell culture and an aliquot of thetreated sample (either virus or infected cell extract) under conditionsthat would result in infection of the deficient cell culture if anyvirus was present in the treated sample. After exposure to the treatedsample, the deficient indicator cell culture is cultured further andassayed for the replication of the virus, by standard method (forexample, plaque assays or TCID₅₀ assays or Northern or Western analysisfor viral RNA or protein).

The host cell culture may be any cell culture which is susceptible toinfection by the virus against which the antiviral compound is to betested. The indicator cell culture is a PKR-deficient or 2-5A synthetasedeficient cell culture that is used to assay for infectious virusremaining after treatment with the antiviral compound. The indicatorPKR-deficient or 2-5 A synthetase deficient cell culture is prepared asdescribed above for vaccine production. Cells suitable as a parent forgenerating the deficient indicator are the same as those that are usefulfor generating the PKR-deficient or 2-5A synthetase deficient cellcultures for vaccine production. In addition, the following cell linesare also suitable: hepatoma cell lines in general, particularly Hep G2human hepatocellular carcinoma (Nature 1979 282:615-616; U.S. Pat. No.4,393,133) and Hep 3B (U.S. Pat. No. 4,393,133). It will be apparentthat the indicator cell culture is also susceptible to infection by thevirus against which the antiviral compound is to be treated. The hostcell culture and the indicator cell culture may be the same ordifferent. The antiviral compound can be any chemical or biologicalpreparation suspected of having some antiviral activity. If the virusitself is treated with the antiviral compound, the compound may beremoved before infection of the indicator cell culture by exposure tothe treated virus. If an infected host cell culture (or a pre-infectedhost cell culture) is treated with the antiviral compound, the compoundmay be removed before preparation of the cell extract.

In a separate related aspect, the present invention provides a methodfor identification and culture of viral pathogens. The permissiveness ofPKR-deficient cells to viral replication makes them particularly usefulin a method to detect very low levels of virus and/or viruses that aredifficult to culture, for example, HIV in monocytes or lymphocytes ofneonates. In this aspect the present invention comprises the steps of(1) exposing under infective conditions a PKR-deficient or a 2-5Asynthetase-deficient cell culture to a sample suspected of containing avirus and (2) assaying for the presence of replicated virus in theexposed cells. The practice of this aspect of the present invention issimilar to that of the previous aspect except that treatment withantiviral compound is omitted. In this aspect, the sample to be assayedfor the presence of virus is generally a clinical sample from a patientsuspected of having a viral infection. The sample may be any appropriateclinical sample including blood, saliva, urine, as well as biopsysamples from lymph node, lung, intestine, liver, kidney and braintissue. The sample may be treated appropriately to release viralparticles (for example, cell extracts may be prepared) or the sample maybe used as received from the patient. The sample or an aliquot of thesample is exposed under infective conditions to a deficient indicatorcell culture and the presence of any replicating virus is determined asdescribed above.

Specific examples of the steps described above are set forth in thefollowing examples. However, it will be apparent to one of ordinaryskill in the art that many modifications are possible and that theexamples are provided for purposes of illustration only and are notlimiting of the invention unless so specified.

EXAMPLES Example 1 Preparation of plasmids

The cDNA inserts corresponding to the wild type human PKR gene and thedominant negative Arg²⁹⁶ !PKR mutant gene, from the plasmids pBS-8.6Rand yex6M (Meurs E, Chong K, Galabru J. et al. Cell 1990 62: 379-90;Chong et al. EMBO J. 11:1553-1562 1992), respectively, were released byHindIII digestion and subcloned into pRC-CMV (Invitrogen), aconstitutive eukaryotic expression plasmid containing a G418-resistancemarker. The orientation of the inserts in selected clones was determinedby restriction digest analysis and confirmed by sequencing (Sequenase2.0, USB). This procedure resulted in the isolation of the expressionplasmids used, pPKR-AS (containing the PKR cDNA in an antisenseorientation under the control of the CMV promoter in the vector) and pArg²⁹⁶ !PKR (containing the Arg²⁹⁶ PKR cDNA under the control of the CMVpromoter in the vector).

Example 2 Isolation of PKR-deficient stable transfectants

Stable transfectants were obtained by electroporation of 5×10⁶exponentially growing U937 cells with 10 μg of each plasmid, inserum-free RPMI-1640 containing DEAE-dextran (50 μg/mL), with a GenePulser apparatus (BioRad) set at 500 μF, 250V. Bulk populations ofstable transfectants were obtained by selection with 400 μg/mL geneticin(GIBCO-BRL) for 3 weeks. Clonal lines were subsequently obtained bylimiting dilution cloning. Cell lines were cultured in RPMI-1640containing 10% fetal calf serum (complete media) and geneticin.

Five representative cell lines were selected for initialcharacterization: "U937-neo" (also called U9K-C) was the control cellline transfected with the parental vector, pRC-CMV; "U937-AS1" (alsocalled U9K-A1) and "U937-AS3" (also called U9K-A3) were independentclones transfected with PPKR-AS; "U937-M13" (also called U9K-M13) and"U937-M22" (also called U9K-M22) were independent clones transfectedwith p Ar²⁹⁶ !PKR.

Example 3 Characterization of PKR-deficient transfectants

PKR kinase activity was measured in an autophosphorylation assay thatuses poly(I):poly(C)-cellulose for binding and activation of PKR enzyme.PKR autophosphorylation assay was performed essentially as described byMaran et al. with the following modifications. Cell extracts (100 μg ofprotein per assay) were incubated with poly(I):poly(C)-cellulose for 1hour on ice, washed three times, and incubated for 30 minutes at 30° C.in 50 μl of a reaction buffer (20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM2-mercaptoethanol, 1.5 mM Magnesium acetate, 1.5 mM MnCl₂) containing 1μCi of -γ-³² P!ATP. Proteins were separated on a 10% SDS-polyacrylamidegel and analyzed by autoradiography.

Cell extracts from IFN-treated HeLa and mouse L929 cells were used aspositive controls, since PKR activity in these cells has been previouslycharacterized (Meurs et al.) (FIG. 1A, lanes 1 and 8). U937-neo cellscontained low basal levels of PKR activity which increased followingtreatment with IFN-α (FIG. 1A, lanes 2 and 3). PKR activity in theparental, untransfected U937 cells was similar to U937-neo cells.However, PKR activity was not detected in any of the four cell linestransfected with pPKR-AS or p Arg²⁹⁶ !PKR plasmids. Furthermore,treatment of these cells with IFN-α did not restore PKR activity (FIG.1A, lanes 4-7), nor did treatment with IFN-γ.

Example 4 Western analysis of PKR-deficient transfectants

To further confirm the inhibition of PKR expression in thepPKR-AS-transfected cell lines, Western blot analysis was performedusing a monoclonal antibody specific for human PKR. Cell extracts (100μg) were separated on a 10% SDS-polyacrylamide gel andelectrotransferred onto nitrocellulose membrane. The membranes wereincubated with anti-PKR monoclonal antibody (Meurs et al. Cell 1990) at1:1000 in BLOTTO (5% nonfat dry milk, 0.05% Tween-20 in Tris-bufferedsaline). Final detection of PKR was facilitated by probing with asecondary horseradish peroxidase-conjugated goat anti-mouse antibody(Santa Cruz Biotech) and using a chemiluminesence method (Amersham ECL).

Basal level of PKR protein was detectable in U937-neo cells (FIG. 1B,lane 1) and increased following treatment with IFN-α or IFN-γ (FIG. 1B,lanes 2 and 3). In contrast, PKR expression was significantly diminishedin both U937-AS1 and U937-AS3 cells (FIG. 1B, lanes 4 and 6) and did notincrease following treatment with IFN-α (FIG. 1B, lanes 5 and 7). WhilePKR protein was detectable in U937-M13 and U937-M22 cells, the mutantArg²⁹⁶ !PKR protein was not distinguishable from wild type PKR by usingWestern blot analysis.

Example 5 Enhanced EMCV replication in PKR-deficient cells

Since the IFN system plays a major role in antiviral responses, weinvestigated whether loss of PKR function would affect the rate ofencephalomyocarditis virus (EMCV) replication. Stocks of EMCV (ATCC No.VR-1314) were prepared by passage in L929 cells. For determination ofEMCV replication, U937-derived transfectants were cultured in completemedia with or without IFNs (recombinant human IFN-α2, Schering;recombinant human IFN-γ, Amgen) for 18 hours. Following two washingswith PBS, the cells were incubated with EMCV in serum-free media for 2hours. The cells were washed again twice and replenished with mediacontaining 1% FCS. Samples were collected at the required time pointsand lysed by three rounds of freeze-thaw. Four-fold serial dilutions ofthe samples were added onto L929 monolayers and incubated for 48 hours,followed by staining with 0.05% crystal violet to determine cytopathiceffects and median tissue culture infective dose (TCID₅₀).

In the control U937-neo cell line following challenge with EMCV at 0.1TCID₅₀ /cell, viral titers peaked at approximately 10⁴ TCID₅₀ /mL after48 hours and did not increase further after 72 hours (FIG. 2A). However,in U937-AS1 and U937-M22 cells, EMCV replication was substantiallyhigher reaching titers of 10⁴ to 10⁵ TCID₅₀ /mL after only 24 hours and10⁸ TCID₅₀ /mL by 48 hours, representing a 10³ to 10⁴ increase in viralyield over that obtained in control cells. In separate experiments usinga lower virus inoculum of 0.001 TCID₅₀ /cell, more dramatic differenceswere observed in EMCV susceptibility between the control and thePKR-deficient cells (FIG. 2B). Under these conditions, EMCV replicationin U937-neo cells was minimal, not exceeding 10² TCID₅₀ /mL even after72 hours, while high viral titers of 10⁸ TCID₅₀ /mL were attained after48 hours in both U937-AS1 and U937-M22 cells. The results indicated thatby suppressing PKR activity in vivo, the cells become very permissive toviral replication, showing as much as a thousand-fold increase overcontrol cells.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the describedinvention.

What is claimed is:
 1. A method for production of a viral vaccine for ananimal virus comprising:(a) infecting a cell culture with a donor virus,wherein said cell culture is deficient in PKR activity, wherein saidPKR-deficient cells are obtained by a process selected from the groupconsisting of:i. transfection of a parent cell line with a PKR antisensepolynucleotide, ii. unaided uptake into a cell line by culturing saidcell line in the presence of a PKR antisense polynucleotide, iii.transfection of a parent cell line with a PKR dominant negative mutantgene, said mutant gene selected from the group consisting of Arg²⁹⁶!PKR, mutants with deletions between amino acid residues 39-59, mutantswith deletions between amino acid residues 58-69, mutants having amutation at glycine 57, and mutants having a mutation at lysine 60; andiv. culturing a cell line in the presence of 2-aminopurine; (b)culturing said infected cell culture under conditions sufficient toprovide efficient virus growth; and (c) harvesting the virus produced.2. The method of claim 1, wherein said PKR-deficient cells are obtainedby transfection of a parent cell line with a PKR antisensepolynucleotide.
 3. The method of claim 1, wherein said deficient cellculture is obtained by culturing a cell line in the presence of PKRantisense polynucleotides.
 4. The method of claim 1, wherein saiddeficient cell culture is a human cell culture.
 5. The method of claim1, wherein said deficient cell culture is derived from a cell lineselected from the group of MRC-5, WI-38, Chang liver, U937, Vero, MRC-9,IMR-90, IMR-91, Lederle 130, MDCK, H9, CEM, and CD4-expressing HUT78. 6.The method of claim 5, wherein said deficient cell culture is derivedfrom MRC-5 or WI-38 or Vero cells.
 7. The method of claim 1, whereinsaid donor virus is an attenuated virus.
 8. The method of claim 2,wherein said PKR antisense polynucleotide is contained in a vector underthe control of a promoter.
 9. The method of claim 8, wherein saidpromoter is an inducible promoter.
 10. The method of claim 3, whereinsaid PKR antisense polynucleotides are 2'-5'oligoadenylate-linkedpolynucleotides.
 11. The method of claim 1, wherein said harvested virusis purified from cell culture components.
 12. The method of claim 1,wherein said PKR-deficient cells are obtained by transfection of aparent cell line with a PKR dominant negative mutant gene.
 13. Themethod of claim 12, wherein said dominant negative mutant is Arg²⁹⁶!PKR.
 14. The method of claim 1, wherein said deficient cell culture isobtained by culturing a cell line in the presence of 2-aminopurine. 15.The method of claim 1, wherein said deficient cell culture is derivedfrom U937 cells.
 16. The method of claim 1, wherein said donor virus isa recombinant virus.
 17. The method of claim 1, wherein said donor virusis a human virus.
 18. The method of claim 17, wherein said donor virusis a human influenza virus.
 19. The method of claim 1, wherein saiddonor virus is a non-human virus.